TY  - JOUR
AU  - Yu, T.-W.
AU  - Müller, R.
AU  - Müller, M.
AU  - Floss, H. G.
TI  - Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid : the starter unit of rifamycin biosynthesis in Amycolatopsis mediterranei S699
JO  - The journal of biological chemistry
VL  - 276
SN  - 0021-9258
CY  - Bethesda, Md.
PB  - Soc.
M1  - PreJuSER-33246
SP  - 12546 - 12555
PY  - 2001
N1  - Record converted from VDB: 12.11.2012
AB  - To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/d-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-d-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.
KW  - Actinomycetales: enzymology
KW  - Actinomycetales: genetics
KW  - Amino Acid Sequence
KW  - Aminobenzoic Acids: metabolism
KW  - Base Sequence
KW  - DNA Mutational Analysis: methods
KW  - DNA Primers
KW  - Genes, Bacterial
KW  - Genes, Regulator
KW  - Molecular Sequence Data
KW  - Multigene Family
KW  - Open Reading Frames
KW  - Plasmids
KW  - Promoter Regions, Genetic
KW  - Restriction Mapping
KW  - Rifabutin: metabolism
KW  - Rifamycins: biosynthesis
KW  - Aminobenzoic Acids (NLM Chemicals)
KW  - DNA Primers (NLM Chemicals)
KW  - Rifamycins (NLM Chemicals)
KW  - Rifabutin (NLM Chemicals)
KW  - 3-amino-5-hydroxybenzoic acid (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:11278540
UR  - <Go to ISI:>//WOS:000168198600013
DO  - DOI:10.1074/jbc.M009667200
UR  - https://juser.fz-juelich.de/record/33246
ER  -